Efficient fluorography of 3H and 14C on thin layers.

Three methods for efficient detection of ³H and ¹⁴C on thin layers are described. The first method uses 0.4% PPO dissolved in 2-methylnaphthalene. It is approximately 15-fold more efficient than 7% PPO-ether for ³H detection and 15-fold more efficient than autoradiography for ¹⁴C (or ³⁵S) detection. The second method uses PPO dissolved in ether. Efficiency of ³H detection increases as the PPO concentration is increased. The third method uses melted PPO and is as efficient as the first method when the film is exposed at ?78°C.     

Sensitive automated methods for phosphate and (Na+ plus K+)-ATPase

Sensitive automated methods for phosphate and (Na⁺ + K⁺)-ATPase are presented. The automated systems use sampler and pump modules from a Technicon autoanalyzer along with a Gilford spectrophotometer. The phosphate assay has a molar absorbance of approximately 87,000 m^?1 cm^?1 640 nm. The method uses a single color reagent and has been used successfully with perchloric acid digests of phospholipids.The (Na⁺ + K⁺)-ATPase method is based on the phosphate method. The method is comparable to manual methods in the amount of ATP and enzyme used per assay. The blank due to ATP is low. The precision of the assay on replicate samples is usually within ±1%. The total time delay for a single assay is less than 9 min and the method can be operated at a rate of 30 samples/hr. The method has been particularly useful in enzyme purification work. Procedures for the use of the method in kinetic studies are described     

A Novel Method for Relative Quantitation of N-Glycans by Isotopic Labeling Using 18O-Water

Quantitation is an essential aspect of comprehensive glycomics study. Here, a novel isotopic-labeling method is described for N-glycan quantitation using ¹⁸O-water. The incorporation of the ¹⁸O-labeling into the reducing end of N-glycans is simply and efficiently achieved during peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase F release. This process provides a 2-Da mass difference compared with the N-glycans released in ¹⁶O-water. A mathematical calculation method was also developed to determine the ¹⁸O/¹⁶O ratios from isotopic peaks. Application of this method to several standard glycoprotein mixtures and human serum demonstrated that this method can facilitate the relative quantitation of N-glycans over a linear dynamic range of two orders, with high accuracy and reproducibility.     

Determination of the specific radioactivity of 14C-labeled glutamic acid and glutamine

A new, simple, and accurate method for the sequential determination of the specific radioactivity of [1-¹⁴C]glutamic acid and [1-¹⁴C]glutamine is described. Using this method, radioactivity in H¹⁴CO₃^?, in [¹⁴C]glutamic acid, and in [¹⁴C]-glutamine can be readily determined on a single sample of blood plasma. Radioactivity is released as ¹⁴CO₂ in a stepwise fashion, trapped in the center wells, and counted in a liquid scintillation counter. The applicability of the method is discussed.     

Measurement of the cytosolic sodium ion concentration in rat brain synaptosomes by a fluorescence method

A method for the measurement of the cytosolic Na⁺ concentration in intact synaptosomes is described. This method makes use of a pH sensitive dye (BCECF) that can be loaded into the cytosol and a relatively specific ionophore (monensin) that can exchange Na⁺ for H⁺ across the synaptosomal membrane. By setting conditions such that there is no electrochemical potential difference for H⁺ across the membrane (no membrane potential and pH_i = pH_o), addition of ionophore would induce a H⁺ flux only if there is a concentration difference for Na⁺. Thus, when there is no fluorescence change (no cytosolic pH change) extracellular [Na⁺] equals intrasynaptosomal [Na⁺. The intrasynaptosomal [Na⁺] concentration was determined to be 7 ± 3 mM (n = 5; mean ± S.E.). The results obtained with this fluorescence method are compared with estimates obtained by atomic absorption spectrometry. Limitations and applications of the method are discussed.     

Improved Most-Probable-Number Method To Detect Sulfate-Reducing Bacteria with Natural Media and a Radiotracer

A greatly improved most-probable-number (MPN) method for selective enumeration of sulfate-reducing bacteria (SRB) is described. The method is based on the use of natural media and radiolabeled sulfate (³⁵SO₄²⁻). The natural media used consisted of anaerobically prepared sterilized sludge or sediment slurries obtained from sampling sites. The densities of SRB in sediment samples from Kysing Fjord (Denmark) and activated sludge were determined by using a normal MPN (N-MPN) method with synthetic cultivation media and a tracer MPN (T-MPN) method with natural media. The T-MPN method with natural media always yielded significantly higher (100- to 1,000-fold-higher) MPN values than the N-MPN method with synthetic media. The recovery of SRB from environmental samples was investigated by simultaneously measuring sulfate reduction rates (by a ³⁵S-radiotracer method) and bacterial counts by using the T-MPN and N-MPN methods, respectively. When bacterial numbers estimated by the T-MPN method with natural media were used, specific sulfate reduction rates (qSO₄²⁻) of 10⁻¹⁴ to 10⁻¹³ mol of SO₄²⁻ cell⁻¹ day⁻¹ were calculated, which is within the range of qSO₄²⁻ values previously reported for pure cultures of SRB (10⁻¹⁵ to 10⁻¹⁴ mol of SO₄²⁻ cell⁻¹ day⁻¹). qSO₄²⁻ values calculated from N-MPN values obtained with synthetic media were several orders of magnitude higher (2 × 10⁻¹⁰ to 7 × 10⁻¹⁰ mol of SO₄²⁻ cell⁻¹ day⁻¹), showing that viable counts of SRB were seriously underestimated when standard enumeration media were used. Our results demonstrate that the use of natural media results in significant improvements in estimates of the true numbers of SRB in environmental samples.     

Simple and rapid quantitative assay of C-labelled urea in human serum using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A simple and rapid quantitative method for ¹³C-labelled urea ([¹³C]urea) in human serum was developed by using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS). This method is used to establish and normalize the [¹³C]urea breath test, which is considered as an effective diagnostic method for Helicobacter pylori infection. HPLC-APCI-MS, involving a simple pretreatment process such as diluting serum with water, was shown to be able to discriminate the extrinsic [¹³C]urea from intrinsic urea present at high concentration in serum. In addition, a ¹³C nuclear magnetic resonance spectroscopic quantitative method for [¹³C]urea in human urine is also described. The precision and accuracy of measured concentrations in these two methods were found to be within the acceptable limit. An application of these methods to investigate the pharmacokinetic profile of orally administered [¹³C]urea in human serum and urine is also presented.     

High-Throughput Measurement and Classification of Organic P in Environmental Samples

Many types of organic phosphorus (P) molecules exist in environmental samples¹. Traditional P measurements do not detect these organic P compounds since they do not react with colorimetric reagents^2,3. Enzymatic hydrolysis (EH) is an emerging method for accurately characterizing organic P forms in environmental samples^4,5. This method is only trumped in accuracy by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy (³¹P-NMR) -a method that is expensive and requires specialized technical training⁶. We have adapted an enzymatic hydrolysis method capable of measuring three classes of phosphorus (monoester P, diester P and inorganic P) to a microplate reader system⁷. This method provides researchers with a fast, accurate, affordable and user-friendly means to measure P species in soils, sediments, manures and, if concentrated, aquatic samples. This is the only high-throughput method for measuring the forms and enzyme-lability of organic P that can be performed in a standard laboratory. The resulting data provides insight to scientists studying system nutrient content and eutrophication potential.     

Determination of Δ-tetrahydrocannabinol levels in brain tissue using high-performance liquid chromatography with electrochemical detection

Δ⁹-Tetrahydrocannabinol (Δ⁹-THC) is the major psychoactive component of cannabis. To assist in investigating the mechanism(s) of action of Δ⁹-THC, a convenient method for determining its levels in brain tissue is required. We now describe a method for determining nanogram quantities of Δ⁹-THC in rat brain tissue. The method employs solvent extraction with methanol-hexane-ethyl acetate, followed by analysis using liquid chromatography with electrochemical detection. Overall recoveries were greater than 80%. The relationship between the peak-height ratio for processed standards extracted in the presence of tissue (Δ⁹-THC/internal standard) and the amount of Δ⁹-THC added was shown to be linear within the range of concentrations examined. Quantitative measurements of Δ⁹-THC in different brain regions following the intravenous administration of Δ⁹-THC are presented as examples of the applications of this method.     

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation¹. Due to its capacity to undergo efficient mitotic homologous recombination, Physcomitrella patens has emerged as an important model system in recent years². This capacity allows high frequencies of gene targeting^3-9, which is not seen in other model plants such as Arabidopsis. To take full advantage of this system, we need an effective and easy method to deliver DNA into moss cells. The most common ways to transform this moss are particle bombardment¹⁰ and PEG-mediated DNA uptake¹¹. Although particle bombardment can produce a high transformation efficiency¹², gene guns are not readily available to many laboratories and the protocol is difficult to standardize. On the other hand, PEG mediated transformation does not require specialized equipments, and can be performed in any laboratory with a sterile hood. Here, we show a simple and highly efficient method for transformation of moss protoplasts. This method can generate more than 120 transient transformants per microgram of DNA, which is an improvement from the most efficient protocol previously reported¹³. Because of its simplicity, efficiency, and reproducibility, this method can be applied to projects requiring large number of transformants as well as for routine transformation.     

Ion chromatographic study of sodium,potassium and ammonium in human body fluids with bulk acoustic wave detection

In the present paper, determination of Na⁺, K⁺ and NH₄⁺ in saliva and serum was carried out using an ion chromatography (IC) method with a bulk acoustic wave (BAW) sensor as detector and 2.0 mmol/l nitric acid as mobile phase. The IC–BAW method is simple, rapid and accurate. Comparison between the BAW detector and the conventional conductivity detector (CDD) was discussed. The IC–BAW showed agreement with the commonly used flame photometric method for Na⁺ and K⁺ and enzymatic method for NH₄⁺, as well as IC–CDD for those three cations.     

High-performance liquid chromatography separation of some NAD+ derivatives suitable for covalent binding

Separation of NAD⁺, N¹-carboxymethyl-NAD⁺, N⁶-carboxymethyl-NAD⁺, and N⁶-[N-(6-aminohexyl)carbamoylmethyl]-NAD⁺ by high performance liquid chromatography is described. Reversed-phase chromatography with the acidic mobile phase (phosphate buffer pH 2.0–3.6) proved to be the most suitable method, particularly for the separation of impurities. The proposed method can be used for monitoring the course of the synthesis of N⁶-[N-(6-aminohexyl)carbamoylmethyl]-NAD⁺ and for the separation of the intermediates. Identification of the peaks was performed by means of spectroscopic measurement as well as a specific coenzyme activity test. Performance of the described method is greater in comparison with thin-layer chromatography.     

Crystal violet–G-quadruplex complexes as fluorescent sensors for homogeneous detection of potassium ion

A novel K⁺ detection method was reported using a label-free G-quadruplex-forming oligonucleotide and a triphenylmethane fluorescent dye crystal violet (CV). This method is based on the fluorescence difference of some CV/G-quadruplex complexes in the presence of K⁺ or Na⁺, and the fluorescence change with the variation of K⁺ concentration. According to the nature of the fluorescence change of CV as a function of ionic conditions, two K⁺ detection modes can be developed. One is a fluorescence-decreasing mode, in which T₃TT₃ (5′-GGGTTTGGGTGGGTTTGGG) is used, and the fluorescence of CV decreases with an increased concentration of K⁺. The other is a fluorescence-increasing mode, in which Hum21 (5′-GGGTTAGGGTTAGGGTTAGGG) is used, and the fluorescence of CV increases with an increased concentration of K⁺. Compared with some published K⁺ detection methods, this method has some important characteristics, such as lower cost of the test, higher concentrations of Na⁺ that can be tolerated, adjustable linear detection range and longer excitation and emission wavelengths. Preliminary results demonstrated that the method might be used in biological systems, for example in urine.     

Measurement of CO(2) Assimilation in Soils: an Experiment for the Biological Exploration of Mars

A method is described for the measurement of ¹⁴CO₂ assimilation by microorganisms in soils. A determination involves exposing soil to ¹⁴CO₂, pyrolyzing the exposed soil, trapping the organic pyrolysis products on a column of firebrick coated with CuO, combusting the trapped organics by heating, and measuring the radioactivity in the CO₂ produced in the combustion. The detection of significant levels of ¹⁴C in the trapped organic fraction appears to be an unambiguous indication of biological activity. The ¹⁴CO₂ which is adsorbed or exchanged into soils by nonbiological processes does not interfere. The method easily detects the ¹⁴CO₂ fixed by 10² to 10³ algae after light exposure for 3 to 24 hr. Assimilation of ¹⁴C is also demonstrable in dark-exposed soils containing 10⁵ to 10⁶ heterotrophic bacteria. Possible applications of the method in the biological exploration of Mars are discussed.     

13C-Labeled Gluconate Tracing as a Direct and Accurate Method for Determining the Pentose Phosphate Pathway Split Ratio in Penicillium chrysogenum

In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-¹³C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus Penicillium chrysogenum. For comparison, a ¹³C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of P. chrysogenum. For the first time mass isotopomer measurements of ¹³C-labeled primary metabolites are reported for P. chrysogenum and used for a ¹³C-based MFA. Estimation of the PPP split ratio of P. chrysogenum at a growth rate of 0.02 h⁻¹ yielded comparable values for the gluconate tracer method and the ¹³C-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the ¹³C-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the ¹³C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio.     

Radioautography of multiple isotopes using color negative films.

An autoradiographic method using color negative films is described which permits differentiationof common radioisotopic decays (³H, ¹⁴C, ³⁵S, ³²P, and ¹²⁵I) according to the volor of the radiographic image produced. The method allows simultaneous quantitation of ³H and any one of the other isotopes. By this means the need for multiple films or exposures, registration of images, and selective attenuation of the decay particles is eliminated.     

Application of the change in partition coefficient with pH to the structure determination of alkyl substituted guanosines.

A versatile method for the determination of the ionization of guanosine is described. Suitably derivatized alkyl-guanosines are partitioned between organic solvents and aqueous buffer solutions at various pH values. Ionization is revealed by a change in partition coefficient with pH. The method is ideally suited for application to micro samples since any quantitative method can be used to determine the partition coefficient. A procedure for distinguishing N¹ or O⁶, N², and C⁸ alkylation of guanosine is described.     

An easy and rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts obtained from in vitro rat heart preparations labeled with 32Pi

A rapid method for the measurement of [γ-³²P]ATP specific radioactivity in tissue extracts containing other ³²P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of ³²P into casein from [γ-³²P]ATP is measured by perchloric acid precipitation of the protein on filter paper. ³²P-Casein formation is linearly related to the specific radioactivity of the [γ-³²P]ATP. Separation of ATP from other ³²P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-³²P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to ³²P_i is demonstrated.     

Double labeling of chromatin proteins,in vivo and in vitro,and their two-dimensional electrophoretic resolution

A modification of the two-dimensional electrophoretic method that involves nonequilibrium pH gradients has been adapted for high resolution of chromatin proteins from sea urchin embryos. A simple method of labeling the protein, in vitro, by reductive methylation with boro[³H]hydride to a specific activity of 100,000 cpm/μg of protein is detailed. Chromatin protein may be labeled, in vivo with ¹⁴C-amino acids, and newly synthesized (³H and ¹⁴C-labeled) and preexistent proteins (only ³H labeled) may be distinguished. The method reveals that sea urchin embryo chromatin contains over 200 proteins.     

Double-label autoradiography on polyacrylamide gels with 3H and 14C

A method of autoradiography is deseribed which allows discrimination between ³H- and ¹⁴C-labeled materials on polyacrylamide gels. The method relies on a very simple procedure for switching from fluorography to autoradiography which results in a decrease in ¹⁴C detection sensitivity of about 10-fold but a concomitant decrease in ³H sensitivity of over 800-fold. With the proper ratio of ³H to ¹⁴C radioactivities, there is little or no “spillover” of one isotope during the detection of the other. No special equipment is required.